A growing body of evidence suggests that dopamine receptors (DARs) do not exist as singular independent units, but rather are part of a large macromolecular complex of interacting proteins. To determine the complement of proteins that make up this DAR signalplex, we have employed a co-immunoprecipitation assay for DARs coupled with mass spectrometry (MS) sequencing. Interacting proteins identified through these experiments include the Na+/K+-ATPase (Na+ pump, NKA), a finding confirmed by both Western analysis and reverse co-immunoprecipitation experiments, which revealed specific D1 and D2 DAR interactions with the NKA &#945;1 subunit. The NKA is a transmembrane protein consisting of both &#945; and &#946; subunits, with &#945;1 being the predominant &#945; isoform. Studies indicate that the &#945; subunit is primarily responsible for the transport of Na+ and K+ across the plasma membrane, while the &#946; subunit may be necessary for targeting of the &#945; subunit. To determine the impact of NKA on DAR function, biological assays were conducted in the presence of enhanced levels of NKA &#945;1 in HEK293T cells. In this system, over-expression of NKA &#945;1 dramatically decreased total D1 and D2 DAR number, with a concomitant functional decrease in DAR-mediated regulation of cAMP production. Interestingly, pharmacological inhibition of either over-expressed or endogenous NKA with ouabain produced an increase in DAR activity, as measured by cAMP production, apparently by disrupting the DAR-NKA interaction. Similarly, biochemical inhibition of endogenous NKA &#945;1 subunit with siRNA also enhanced DAR function. In addition, NKA function was assessed following over-expression of DARs. These experiments revealed that interaction with DARs under basal conditions decreases endogenous NKA activity, while dopamine stimulation of the D1 receptor enhances NKA function. These data indicate that the DARs and NKA can reciprocally regulate one another, providing a control mechanism for both DAR signaling and cellular ion balance. Current studies are underway to determine both the localization of these DAR-NKA complexes and the consequence of receptor stimulation or desensitization on DAR-NKA interactions. The role of the NKA &#946; subunit and whether this reciprocal regulation is mediated by direct or indirect interactions are also being investigated.[unreadable] [unreadable] As noted above. dopamine receptors (DARs) do not exist as singular independent units within the synaptic membrane, but are part of large macromolecular complexes of interacting proteins. We have further employed a co-immunoprecipitation assay for DARs from transfected cell lines, coupled with mass spectrometry sequencing to identify interacting partners. Through these studies we have identified sorting nexin-25 (SNX25) as a DAR interacting protein. Mammalian SNXs have been suggested to be involved in intracellular trafficking, internalization, and endosomal recycling or sorting. Thus far, 27 SNXs have been identified in humans, all defined by the presence of the phox (PX) domain. SNX25 also contains an RGS (regulator of G-protein signaling) domain. The physiological role of SNX25 is unknown. When SNX25 is over-expressed with either D1 or D2 DARs in HEK293 cells, the receptors demonstrate increased expression levels as determined by radioligand binding and cAMP functional assays. There were no effects of increasing SNX25 levels on basal or forskolin-stimulated cAMP levels in the cells. This increase in receptor number was accompanied by an alteration in cellular distribution of the receptors to small internal compartments as seen using confocal microscopy. Intact cell binding assays using the D2 DAR confirmed this change in surface localization. This altered receptor distribution to internal compartments was not a result of receptor degradation as these compartments were not found in lysosomes as determined with co-localization studies using a lysosomal marker. The precise location of these intracellular clusters is a focus of current studies. We further examined the influence of SNX25 on DARs by blocking the expression of endogenous SNX25 using a plasmid based siRNA approach. These studies found that decreasing the amount of SNX25 results in a decrease in both D1 and D2 DAR expression. We also examined the influence of SNX25 over-expression on D1 DAR desensitization and found that SNX25 over-expression does not appear to effect D1 DAR down-regulation via desensitization. Overall, these data suggest that SNX25 regulates the intracellular trafficking of D1 and D2 DARs, the precise mechanism is being investigated.